Project Summary The projects proposed for this grant application seek to test novel transplant (HCT)-based immunomodulatory combination treatments for AML and MDS, to dissect how leukemia cells and their surrounding immune cell populations co-evolve in relationship to allo-HCT course (with the aim of gaining essential insights into the rational design of effective combination therapy for AML and MDS), and to understand how donor clonal hematopoiesis (CHIP) in the stem cell population impacts the reconstitution of immune cell subpopulations. Core 3 will support the single cell- and immunogenomics-related goals of these projects by focusing on applying the latest computational and experimental tools to these studies. Core 3 will analyze whole exome sequencing data from leukemia and normal samples to identify cancer-specific somatic mutations and polymorphic differences between donor and recipient, and use matched RNA sequencing data to determine which of these variants are expressed. Recently developed sophisticated algorithms will be implemented to use this information to predict personal HLA-binding peptides that compose personal leukemia neoantigens and hematopoietic-lineage restricted minor histocompatibility antigens (Aim 1). In order to determine the immune response to HCT, single- cell transcriptome sequencing of non-tumor immune cell populations will be used to identify pathways related to immune cell functions (Aim 2). Targeted RNA analysis will be used to determine how these discovered phenotypes relate to the genotype of individual cells and will be used to define how CHIP affects response to HCT. Response of T cells to HCT will be further characterized by TCR repertoire analysis using targeted bulk and single-cell sequencing to assess T cell clonality (Aim 3). The paired alpha/beta TCR chain single-cell sequence information will be used to reconstruct cell lines expressing individual enriched TCRs (Aim 4) in order to functionally determine exactly which TCR interacts with which antigen. This analysis will directly assess if neoantigen- or mHAg-directed T cell responses contribute to clinical responses to therapy.